A large number of clinically and biologically significant substances (e.g. enzymes) can be quantifiably determined using a reaction or sequence of reactions that generate a detectable reductant such as nicotinamide adenine dinucleotide, reduced form (hereinafter NADH) or nicotinamide adenine dinucleotide phosphate, reduced form (hereinafter NADPH). Most conventional assay methods which monitor either NADH or NADPH do so by directly measuring either the change in absorbance due to nicotinamide adenine dinucleotide (hereinafter, NAD) or nicotinamide adenine dinucleotide phosphate (hereinafter, NADP) when they are reduced, or by measuring the fluorescence change which occurs with that reduction.
These conventional procedures, however, present a number of problems. They are generally highly pH-sensitive and subject to considerable error if strict pH control is not maintained. Further, NADH and NADPH are relatively unstable compounds. Also, the sensitivity of such methods is relatively low due to the low extinction of NADH. These methods require the use of complicated optical equipment capable of operating in the ultraviolet region of the electromagnetic spectrum and are subject to interferences which arise in that region.
To improve the sensitivity of such assays and to avoid the problems with UV measurements, NAD or NADP reduction has been used with tetrazolium salts to yield formazan dyes. However, formazan dyes also have generally low extinction coefficients and the tetrazolium structures cannot be readily modified to increase the extinction.
U.S. Pat. No. 3,331,752 (issued July 18, 1967 to Struck, Jr. et al) describes a solution assay for enzymes which can be reacted to produce NADH. This assay uses a ferric ion chelate which is reduced by the NADH to form the corresponding ferrous ion chelate which has different absorption characteristics than its ferric ion counterpart. The ligand is the same material for both the ferric and ferrous chelates. This assay has a serious drawback, however. It is laborious and time-consuming since it requires a number of steps for successful completion. For example, enzyme reaction and NADH formation is carried out in solution at a suitable pH. After a desired time (5-20 minutes), the pH is changed by adding an acid or base to stop the enzyme reaction and resulting NADH formation. Simultaneously or subsequently to this, the ferric ion chelate is added to the mixture, and after another lengthy period of time, the resulting change in color of the ferrous ion chelate is measured. This laborious procedure is susceptible to inaccurate results and is not suitable for highly automated clinical procedures.
A triglycerides assay is described in U.S. Pat. No. 4,245,041 (issued Jan. 13, 1981 to Denney). In this assay, free ferric ions, an electron transfer agent, suitable triglyceride reagents and a ferrous ion chelating agent are mixed in a solution with the liquid sample. In the presence of triglycerides, the reaction sequence produces NADH which then reduces the ferric ions to ferrous ions which coordinate with the chelating agent. The resulting ferrous ion chelate is a colored dye which is easily detected. However, this assay presents a problem because uncomplexed ferric ions remaining in the composition are gradually reduced by undetermined reductants in the assay environment. Hence, the amount of ferrous ion chelate dye formed increases with time, causing inaccurate determinations. Further, if reagent blanks are used in calibrating the spectrophotometer used to measure the dye, the blanks can change with time and the instrument will have to be calibrated frequently. Clearly these disadvantages make this assay undesirable in view of the need for rapid, inexpensive and reliable assays in clinical laboratories today.
The problem of ferric ion instability in the triglycerides assay of Denney was noted in Japanese Patent Publication 56-153961 (published Apr. 4, 1983). This reference teaches the addition of a ferric ion masking agent (i.e. chelating agent) to the assay solution to complex with excess ferric ions and to allegedly arrest undesirable ferric ion reduction. This addition occurs after the assay for the analyte has been completed. Use of such a masking agent, however, requires that the assay conditions be carefully tailored so that the masking agent will chelate with the ferric ions. For example, the chelation is highly pH-dependent. So, the assay pH and the chelating agent pH must correspond in order for successful masking to occur, or the pH has to be changed when the masking agent is added. This procedure requires an additional step of adding the masking agent after dye formation, thereby complicating the assay and making it less desirable for highly automated analytical procedures.
In general, the ferric ion chelates described in the art are formed from weak chelating agents. Therefore, the ferrous ion chelating agents readily complex also with ferric ions, thereby making the ferric ions more easily reducible by reductants that may be present as interferents.
In view of the problems noted above with known assays, it would be desirable to have a simple, rapid and reliable assay for determining NADH, NADPH or similar reductants in highly automated analytical procedures.